ABSTRACT
As a class of multifunctional biocatalysts, halohydrin dehalogenases are of great interest for the synthesis of chiral β-substituted alcohols and epoxides. There are less than 40 halohydrin dehalogenases with relatively clear catalytic functions, and most of them do not meet the requirements of scientific research and practical applications. Therefore, it is of great significance to excavate and identify more halohydrin dehalogenases. In the present study, a putative halohydrin dehalogenase (HHDH-Ra) from Rhodospirillaceae bacterium was expressed and its enzymatic properties were investigated. The HHDH-Ra gene was cloned into the expression host Escherichia coli BL21(DE3) and the target protein was shown to be soluble. Substrate specificity studies showed that HHDH-Ra possesses excellent specificity for 1,3-dichloro-2-propanol (1,3-DCP) and ethyl-4-chloro-3-hydroxybutyrate (CHBE). The optimum pH and temperature for HHDH-Ra with 1,3-DCP as the reaction substrate were 8.0 and 30 °C, respectively. HHDH-Ra was stable at pH 6.0-8.0 and maintained about 70% of its original activity after 100 h of treatment. The thermal stability results revealed that HHDH-Ra has a half-life of 60 h at 30 °C and 40 °C. When the temperature is increased to 50 °C, the enzyme still has a half-life of 20 h, which is much higher than that of the reported enzymes. To sum up, the novel halohydrin dehalogenase from Rhodospirillaceae bacterium possesses good temperature and pH stability as well as catalytic activity, and shows the potential to be used in the synthesis of chemical and pharmaceutical intermediates.
Subject(s)
Escherichia coli/metabolism , Hydrolases/metabolism , Rhodospirillaceae , Substrate SpecificityABSTRACT
2-Haloacid dehalogenases (EC 3.8.1.X) catalyze the hydrolytic dehalogenation of 2-haloacids, releasing halogen ions and producing corresponding 2-hydroxyacids. The enzymes not only degrade xenobiotic halogenated pollutants, but also show wide substrate profile and astonishing efficiency for enantiomer resolution, making them valuable in environmental protection and the green synthesis of optically pure chiral compounds. A variety of 2-haloacid dehalogenases have been biochemically characterized so far. Further studies have been made in protein crystal structures and catalytic mechanisms. Here, we review the recent progresses of 2-haloacid dehalogenases in their source, protein structures, reaction mechanisms, catalytic properties and application. We also suggest further research directions for 2-haloacid dehalogenase.
Subject(s)
Catalysis , Halogenation , Hydrolases , Chemistry , Metabolism , Hydrolysis , Research , Substrate SpecificityABSTRACT
No Brasil, estima-se que as intoxicações por plantas tóxicas que contém monofluoroacetato de sódio (MFA) causam a morte de aproximadamente 500.000 bovinosao ano. A inoculação ruminal de bactérias que degradam MFA tem sido proposta como uma forma de prevenir a intoxicação. O presente trabalho teve como objetivo avaliar, em caprinos, a resistência ao MFA presente em Amorimia septentrionalis, induzida por inoculação ruminal das bactérias Pigmentiphaga kullae e Ancylobacter dichloromethanicus. Doze caprinos, que nunca tiveram contato prévio com plantas que contêm MFA, foram divididos em dois grupos, com seis animais cada. No grupo 1, 60 mL de uma mistura das duas bactérias foi inoculada, diariamente, durante 10 dias em cada caprino. No grupo 2, os caprinos não receberam as bactérias. A partir do 10º dia de inoculação, A. septentrionalis foi administrada, diariamente, na dose de 5g/kg de peso vivo, sendo interrompida em cada animal após a observação dos primeiros sinais clínicos da intoxicação. Os caprinos do grupo 1 apresentaram sinais clínicos 5,83±2,56 dias após a administração da planta o que diferiu significativamente (p=0,037) dos caprinos do grupo 2, que apresentaram sinais clínicos aos 2,67±0,52 dias. A quantidade de planta ingerida pelos caprinos inoculados (28,83±12,97g/kg) e os não inoculados (12,03±3,65g/kg) para desencadear os sinais clínicos foi, também, estatisticamente diferente entre os grupos (p=0,025). Conclui-se que a administração intraruminal de Pigmentiphaga kullae e Ancylobacter dichloromethanicus induz resistência à intoxicação por plantas que contêm MFA.
In Brazil is estimated that poisoning of livestock by sodium monofluoroacetate (MFA) containing plants causes the death of about 500.000 cattle per year. The ruminal inoculation of bacteria that degrade MFA has been proposed as a way to prevent the poisoning. This study aimed to evaluate in goats resistance to the MFA-containing plant Amorimia septentrionalis induced by ruminal inoculation of the bacteria Pigmentiphaga kullae and Ancylobacter dichloromethanicus. Twelve goats, without previous contact with MFA-containing plants, were divided into two groups of six animals each. In group 1, 60ml of a mixture of the two bacteria was inoculated every day for 10 days into each goat. In group 2, the goats did not receive the bacteria. At the 10th day of inoculation, A. septentrionalis began to be administered daily at a dose of 5g/kg body weight to both groups. The administration was interrupted in each goat after first clinical signs of poisoning were observed.. The goats of group 1 showed clinical signs 5.83±2.56 days after the administration of the plant, what differed significantly (p=0.037) from goats of group 2, that showed clinical signs 2.67±0 52 days after the beginning of ingestion. The amount of A. septentrionalis ingested by inoculated goats (28.83±12.97g/kg) to cause clinical sings was significantly greater (p=0.025) than the amount ingested by the non-inoculated (12.03±3.65) goats to cause clinical signs and was also statistically different between the groups. We concluded that the intraruminal administration of Pigmentiphaga kullae and Ancylobacter dichloromethanicus increases the resistance to poisoning by MFA-containing plants.